Virological

Communication

Biosci. Biotech. Res. Comm. 11(2): 300-306 (2018)

A comparative analysis of overall codon usage pattern

of Louping Ill virus with natural livestock host and

associated vector

Anjusha Mune

*, Ajay Pandey

and Khushhali Menaria Pandey

Department of Biological Science and Engineering, MANIT Bhopal (M.P.) India

Department of Applied Mechanics, MANIT Bhopal (M.P.) India

ABSTRACT

Louping ill is a zoonotic viral disease caused by louping ill virus (LIV) which is a member of genus Flavivirus in the

family Flaviviridae. This febrile illness to livestock can further develop into fatal encephalitis .The virus LIV is closely

related to tick-borne encephalitis virus and occurs wherever the primary vector tick (Ixodes ricinus) is found. To

understand the viral evolution, comparison and analysis of the codon usage of LIV, its vector, and the host is impor-

tant. The present study reports the pattern of codon usage in LIV, its vector, and the host by calculating the Effective

number of Codons (ENC), Codon Adaptation Index (CAI), and Relative Synonymous Codon Usage (RSCU) and other

indicators. The results indicate relatively low codon usage bias of LIV. The ENC - plot demonstrates the substantial

role played by mutation pressure. The com parative analysis of CAI among virus, vector and its host, indicates that

the virus is more adaptive to the host than the vector. A comparative analysis of RSCU between virus, vector, and its

host shows that the codon usage pattern of LIV is a mix of coincidence and antagonism. To the best of our knowledge,

this is the  rst report describing codon usage analysis of LIV and  ndings are expected to increase our understanding

of factors involved in viral evolution and  tness toward vector and host.

KEY WORDS: CODON USAGE, EVOLUTION, LOUPING ILL VIRUS (LIV), EFFECTIVE NUMBER OF CODONS, RELATIVE SYNONYMOUS CODON

USAGE

300

ARTICLE INFORMATION:

*Corresponding Author: anjumune@gmail.com

Received 10

March, 2018

Accepted after revision 19

May, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA and

Crossref Indexed Journal

NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at: http//www.bbrc.in/

DOI: 10.21786/bbrc/11.1/16

Anjusha Mune et al.

INTRODUCTION

Louping ill virus (LIV) is a tick-born member of the genus

Flavivirus in Flaviviridae family. It is a positive single

stranded, 40-50 nm RNA virus whose genome comprises

a single open reading frame (ORF) that is approximately

11 kb in length (Grard et al.,2007;Jeffries et al., 2014).

The ORF encodes a polyprotein that consists of three

structural and seven non-structural proteins. The virus

show high degree of genetic homology to tick-borne

encephalitis virus (TBEV) of the same family (McGuire

et al., 1998; Jiang et al., 1993). It is mainly transmitted

by ticks and the primary vector is Ixodes ricinus (Dobler

et al., 2010).LIV mainly causes febrile illness in sheep,

cattle, horse, pigs and some other animals that may

eventually result in fatal encephalitis.

Sheep are the most important reservoir host for LIV.

The disease is dominantly detected in animals from

upland areas of British Isles (Gao et al., 1997) though

the disease is also reported in Scotland, Ireland, and

northern England where the tick vector Ixodes ricinus

is found. Infection with LIV was  rst reported in sheep

of Basque region of northern Spain in 1987 (Gonzalez

et al., 1987). Most of the cases of LI infection occur

in spring / early summer when ticks are common. In

endemic areas morbidity and mortality depends upon

animal’s immune status, concurrent infection and other

factors. All age group of animal get infected by it and

once encephalitis is developed the case fatality rate goes

up to 50%. The mortality rate is even higher in animals

that are less than two years old. Currently, there is no

speci c treatment for LIV with only supportive therapies

being helpful to some extent (Hyde et al., 2007 Mans-

 eld et al., 2015 Butt et al., 2016).

The molecular sequence data started to be accu-

mulated nearly 20 years ago. It was observed that the

genetic code is redundant and most amino acids can be

translated by more than one codon (Wang et al., 2011).

This redundancy is a key factor regulating the ef ciency

and accuracy of protein production.Alternative codons

within the same group that encode the same amino acid

are often called ‘synonymous’ codons. These codons are

not randomly selected within and between genomes.

This is referred to as ‘codon usage bias’ (CUB). CUB are

widespread across the tree of life and are in uenced by

mutation pressure, natural or translational selection,

secondary protein structure, replication, selective tran-

scription, hydrophobicity and hydrophilicity of the pro-

tein, and the external environment (Xiang et al., 2015

Butt et al., 2016 Mune et al., 2017).

As viruses are intracellular pathogens they have to

co-evolve with host molecular mechanisms. The inter-

play between the codon usage of the virus and its host is

expected to affect the overall viral survival,  tness, evasion

of the host immune system and evolution. The knowledge

of the codon usage of viruses can provide information

about their molecular evolution and extend our under-

standing of the regulation of viral gene expression. This

may also offer signi cant improvement in vaccine design

for which the ef cient expression of viral proteins may be

required to generate immunity (Tao et al., 2009 Velazquez

et al., 2016). To gain insight into the characteristics of the

viral genome and evolution, the codon usage patterns of

the three components of transmission cycle, namely - the

virus (LIV), vector (Ixodes ricinus), and hosts (Sheep (Ovis

aries), Pig (Sus scrofa) and cattle (Bos taurus)) were inves-

tigated in our study.

MATERIALS AND METHODS

SEQUENCE DATA

The complete genome sequences were downloaded from

the National Centre for Biotechnology (NCBI) data-

base (http: //www.ncbi.nlm. nih.gov) in FASTA format.

The detailed information (accession numbers, coun-

try, sequence length etc.) of the selected genomes were

listed [Table. S1]. Open reading frames (ORF) of all the

genomic sequences were identi ed by using NCBI ORF

 nder (https://www.ncbi.nlm.nih.gov/orf nder/). The

host (Ovis aries, Sus scrofa and Bos taurus) and vec-

tor (Ixodes ricinus) codon usage were obtained from the

Codon Usage Data Base (CUD).

CODON USAGE ANALYSIS

The overall frequency of occurrence of the nucleotides

(A %, C %, U %, and G %) was calculated along with

the frequency of each nucleotide at the third site of the

synonymous codons (A

, C

, U

and G

).Also the overall

GC, AU and GC

content were calculated using MEGA7

software to investigate the compositional properties of

coding region of LIV. To investigate the codon usage

pattern, the RSCU (Relative synonymous codon usage)

values for synonymous codons were calculated accord-

ing to the published equation (Sharp et al., 1986). The

stop codons (UAA, UAG and UGA) and AUG for Met,

UCG for Try were not introduced into the RSCU anal-

ysis. Further, ENC (Effective number of codon) values

were calculated to measure the magnitude of codon

usage bias in the coding sequences of viral genome. The

ENC value ranges from 20 (when only one synonymous

codon is chosen by the corresponding amino acid) to 61

(when all synonymous codons are used equally). A low

ENC value indicates a strong codon usage bias (Wright

et al., 1990; Zhang et al., 2011 Butt et al., 2013).

The CAI (Codon adaptation index) was used to esti-

mate the adaptation of LIV to its host and vector codons.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS A COMPARATIVE ANALYSIS OF OVERALL CODON USAGE PATTERN OF LOUPING ILL VIRUS WITH 301

Anjusha Mune et al.

CAI values range from 0 to 1. A higher CAI score for a

given gene indicates more similarity between its codon

usage and the prede ned reference set, using the CAIcal

approach (available at: http://genomes.urv.es/CAIcal)

(Puigbo et al., 2008).

RESULTS AND DISCUSSION

SYNONYMOUS CODON USAGE IN LIV

The preference for one type of codon over another can

be greatly in uenced by the nucleotide composition of

genome. We  rst analysed nucleotide composition and

observed that the nucleotides A and G were higher and

followed by C and U (Table 1) The LIV genome is rich

with G content having a mean value of 32.17. For a bet-

ter understanding we analysed nucleotide composition

at third position of codon and observed the dominance

of G

nucleotide with a mean value of 34.20. Even the

percentage of dinucleotide with G is higher compared

to dinucleotide with other nucleotides (respective mean

values for GC, AU, GC

and AU

being 54.74, 45.26,

60.74, and 39.26).

To investigate the extent of codon usage bias, the

ENC values among LIV genome were calculated. An

average value of 53.97 represents stable ENC value

(ENC > 40) (Mune et al., 2017) which suggests that the

genomic composition of LIV is conserved. The result

shows that the codon usage of LIV is slightly biased and

mainly affected by the nucleotide composition. To fur-

ther understand the codon usage pattern, the analysis of

ENC - plot (ENC value V/s GC

content) was carried out.

It is observed that all points lie below the expected curve

(Fig.1). This implies that the codon usage bias is mainly

affected by nucleotide composition (in other words - by

mutation pressure).

To further explore the codon usage preferential opti-

mization and adaptation of LIV in relation to its vector

and hosts CAI analysis was performed. CAI values were

calculating keeping Ixodes ricinus, Ovis aries,Sus scrofa

and Bos taurus codon usage as a reference set. A mean

CAI value of 0.658 was obtained for the LIV ORFs in

relation to primary vector Ixodes ricinus codon usage

reference set and mean CAI values of 0.623, 0.689 and

0.711 were obtained for the LIV ORFs in relation to host

pig , sheep and cattle (Ovis aries,Sus scrofa and Bos tau-

rus) codon usage reference set respectively. In this study

we found a tendency for higher CAI values indicating

lower ef ciency of translation. A comparison between

vector and host indicated a lower CAI for LIV in relation

to pig, which leads to lower ef ciency of protein syn-

thesis in pig. This suggests that the interplay of codon

usage between LIV and its hosts may in uence viral  t-

ness, survival and evolution.

Table 1. Nucleotide composition analysis of LIV genome (IR: Ixodes ricinus, SS: Sus scrofa, OA: Ovis aries, BT: Bos taurus)

Accession no. U C A G U3 C3 A3 G3 AU GC AU3 GC3 GC12 ENC CAI

CAI

NC_001809.1 20.72 22.67 24.47 32.13 18.57 26.53 20.85 34.06 45.19 54.81 39.41 60.59 30.29 53.88 0.658 0.622 0.691 0.711

Y07863 20.72 22.67 24.47 32.13 18.57 26.53 20.85 34.06 45.19 54.81 39.41 60.59 30.29 53.88 0.658 0.622 0.691 0.711

KT224354.1 20.81 22.48 24.57 32.14 18.62 26.47 20.67 34.23 45.38 54.62 39.30 60.70 30.35 54.12 0.657 0.623 0.687 0.711

KP144331.1 20.61 22.59 24.60 32.20 18.16 26.68 20.94 34.23 45.21 54.79 39.09 60.91 30.45 53.89 0.658 0.624 0.689 0.711

KJ495985 20.81 22.48 24.58 32.13 18.62 26.47 20.67 34.23 45.39 54.61 39.30 60.70 30.35 54.15 0.657 0.623 0.687 0.710

KF056331.1 20.74 22.54 24.45 32.27 18.48 26.56 20.59 34.38 45.19 54.81 39.06 60.94 30.47 53.93 0.658 0.622 0.690 0.711

Avg. 20.74 22.57 24.52 32.17 18.50 26.54 20.76 34.20 45.26 54.74 39.26 60.74 30.37 53.97 0.658 0.623 0.689 0.711

Std. D 0.0722 .0890 .0652 .0561 .1780 .0756 .1361 .1234 .0961 .0961 .1532 .1532 .0766 .1261 .0005 .0008 .0018 .0004

302 A COMPARATIVE ANALYSIS OF OVERALL CODON USAGE PATTERN OF LOUPING ILL VIRUS WITH BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Anjusha Mune et al.

FIGURE 2. Comparative analysis of relative synonymous codon usage (RSCU) patterns between virus, vector

and three hosts (cattle, sheep and pig).

FIGURE 1. Graph showing the relationship between the effective number of codons

(ENC) and GC content of the third codon position (GC3).The curve indicates the

expected codon usage if GC compositional constraints alone account for codon

usage bias.

To investigate the codon usage pattern of virus, an RSCU

analysis was performed for the 59 sense codons (Table.2). In

LIV among the 18 most abundantly used codons, 12 were

G/C-ended ( ve G-ended, seven C-ended) and the remain-

ing six were A/U-ended (  ve A-ended and one U-ended).

To determine the potential in uences of the vector

and host on the codon usage pattern of the LIV, the

RSCU pattern of LIV coding sequence were correlated

with those of Ixodes ricinus (vector) and pig, sheep and

cattle (hosts) (Fig.2).All the 18 most abundantly used

codons of vector and host were G/C ending (In Ixodes

ricinus twelve C-ended and six G-ended, Pig thirteen

C-ended and  ve G-ended, cattle twelve C-ended and

six G-ended, and in sheep eleven C-ended codons six

G-ended codons and one U-ended codon) we observed

a common pattern of preference towards G/C-ended

codons in vector and host. An analysis of over and

under - represented codons showed that for LIV 4 out of

18 preferred codons (CUG for Leu, GUG for Val and AGA

and GGA for Arg) in Ixodes ricinus 11 out of 18 preferred

codons (CUG for Leu, AUC for Ile, GUG for Val, AGC for

Ser, CCC for Pro, ACC for Thr, GCC for Ala, CAC for His,

UGC for Cys, AGG for Arg and GGC for Gly), in cattle 3

out18 preferred codons (CUG for Leu, GUG for Val and

GCC for Ala), in sheep 5 out of 18 preferred codons (CUG

and CUC for Leu, AUC for Ile, GUG for Val and ACC for

Thr), and in pig 6 out of 18 preferred codons (CUG for

Leu, AUC for Ile, GUG for Val, AGC for Ser and ACC for

Thr, GCC for Gly) had RSCU value >1.6, whereas the

remaining preferred codons had RSCU values >0.6 and

<1.6. CUG for Leu and GUG for Val are common over-

represented codons in virus vector and hosts.

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Table 2. The relative synonymous codon usage patterns of LIV, its host (cattle,

sheep and pig) and primary transmission vector (Ixodes ricinus)

AA Codon Pathogen Vector Host

louping ill Ixodes ricinus Cattle Sheep Pig

Phe UUU 0.88 0.66 0.85 0.94 0.79

UUC 1.12 1.34 1.15 1.06 1.21

Leu UUA 0.18 0.16 0.38 0.24 0.32

UUG 1.11 0.75 0.71 0.49 0.67

CUU 0.90 1.08 0.7 0.74 0.65

CUC 1.2 1.40 1.26 1.83 1.35

CUA 0.37 0.26 0.36 0.24 0.33

CUG 2.24 2.45 2.59 2.46 2.68

Ile AUU 0.71 0.85 0.98 0.63 0.91

AUC 1.36 1.79 1.57 1.74 1.67

AUA 0.93 0.36 0.45 0.63 0.42

Val GUU 0.7 0.68 0.64 0.46 0.57

GUC 1.1 1.36 1.01 0.91 1.07

GUA 0.29 0.35 0.4 0.36 0.34

GUG 1.92 1.61 1.95 2.27 2.03

Ser UCU 0.69 0.76 1.04 0.91 0.99

UCC 0.81 1.54 1.37 1.28 1.5

UCA 1.11 0.48 0.79 0.48 0.73

UCG 0.64 0.83 0.39 0.28 0.39

AGU 1.17 0.69 0.87 1.48 0.77

AGC 1.58 1.70 1.53 1.58 1.62

Pro CCU 0.96 0.75 1.08 1.26 1.05

CCC 0.98 1.70 1.39 1.29 1.46

CCA 1.36 0.96 1 1.03 0.94

CCG 0.7 0.98 0.53 0.42 0.56

Thr ACU 0.75 0.68 0.89 0.78 0.83

ACC 1.18 1.71 1.55 2.05 1.68

ACA 1.29 0.82 1.01 0.78 0.92

ACG 0.77 1.00 0.56 0.38 0.57

Ala GCU 1.06 1.07 1 1.18 0.96

GCC 1.11 2.69 1.71 1.55 1.8

GCA 1.12 0.84 0.8 0.9 0.74

GCG 0.72 0.95 0.48 0.37 0.5

Tyr UAU 0.61 0.45 0.79 0.72 0.73

UAC 1.39 1.59 1.21 1.28 1.27

His CAU 0.75 0.50 0.75 1.08 0.7

CAC 1.25 1.75 1.25 0.92 1.3

304 A COMPARATIVE ANALYSIS OF OVERALL CODON USAGE PATTERN OF LOUPING ILL VIRUS WITH BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

Anjusha Mune et al.

Gln CAA 0.66 0.60 0.46 0.57 0.44

CAG 1.34 1.16 1.54 1.43 1.56

Asn AAU 0.68 0.55 0.81 0.49 0.79

AAC 1.32 1.07 1.19 1.51 1.21

Lys AAA 0.79 0.65 0.78 0.68 0.76

AAG 1.21 1.04 1.22 1.32 1.24

Asp GAU 0.8 0.54 0.84 0.66 0.8

GAC 1.2 1.40 1.16 1.34 1.2

Glu GAA 0.69 0.91 0.78 0.75 0.72

GAG 1.31 1.02 1.22 1.25 1.28

Cys UGU 1.04 0.57 0.85 0.72 0.79

UGC 0.96 1.62 1.15 1.28 1.21

Arg CGU 0.38 0.75 0.49 0.82 0.44

CGC 0.94 1.59 1.17 1.15 1.31

CGA 0.53 0.80 0.68 0.89 0.6

CGG 0.64 1.04 1.32 0.86 1.29

AGA 1.78 0.83 1.14 1.12 1.12

AGG 1.74 1.62 1.2 1.16 1.23

Gly GGU 0.66 0.78 0.64 0.92 0.57

GGC 0.82 2.01 1.43 1.33 1.46

GGA 1.51 1.31 0.95 1.05 0.91

GGG 1.02 0.67 0.99 0.71 1.05

Supplementary Table 1. Detail information about the LIV

Strain

Name

Virus

Type

GenBank

Accession

Sequence

Length

ORF ORF

Length

Collection

Date

Host GenBank

Host

Country

369/T2 LIV NC_001809 10871 130-10374 10245 -N/A- Unknown -N/A- -N/A-

369/T2 LIV Y07863 10871 130-10374 10245 -N/A- Unknown -N/A- -N/A-

LEIV-7435Tur LIV KT224354 10829 106-10350 10245 -N/A- Tick Hyalomma

marginatum

(tick)

Turkmenistan

LI3/1 LIV KP144331 10880 133-10377 10245 1962 Sheep Ovis aries United

Kingdom

Primorye-

185-91

LIV KJ495985 10871 129-10373 10245 07/22/1991 Human Homo

sapiens

Russia

Penrith LIV KF056331 10875 132-10376 10245 2009 Sheep Ovis aries United

Kingdom

None of the preferred codons were under-represented

(RSCU<0.6). UUA and CUA for Leu and GUA for Val

are common underrepresented codons in virus, vector

and hosts. Interestingly, a mixture of coincidence and

antagonism was observed in the codon usage pattern

as LIV showed no complete coincidence or complete

antagonism to any of the patterns of its vector and host.

Among the 18 most abundantly used codons, the ratio of

coincident/antagonist preferred codon was 12:6 between

virus vector and hosts.

CONCLUSION

Our analysis has provided an insight into codon usage

pattern of LIV virus and its relationship with host and

vector. We observed that the codon usage bias of LIV is

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS A COMPARATIVE ANALYSIS OF OVERALL CODON USAGE PATTERN OF LOUPING ILL VIRUS WITH 305

Anjusha Mune et al.

slightly biased which re ects that the key role played by

mutation pressure and natural selection. Our observa-

tions suggest that codon usage of LIV is an evolutionary

process However, a more comprehensive analysis with

higher sample sizes is needed as this study and sub-

sequent analysis is based on a relatively small sample

size.

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306 A COMPARATIVE ANALYSIS OF OVERALL CODON USAGE PATTERN OF LOUPING ILL VIRUS WITH BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS